The field of immunochemistry, and the development of immunoassay technology, has been evolving since the late 19th century. However, the majority of these methods have been developed for use by the medical community. Immunoassays are based on the highly specific binding between and antibody and the antigen recognized by the antibody. Antibodies are binding proteins that are produced by the immune system of vertebrates in response to substances that are perceived to be foreign.
Various approaches have been described for carrying out immunoassays. The early ELISA's were what is commonly called a "competitive" assay in which the enzyme labeled antigen or antibody competed with the antigen or antibody to be determined for a reaction site on a bead, pad or surface to which one member of an immunologically coupling pair was attached. Subsequently, the "sandwich" assay was developed. In the sandwich assay, the antibody or antigen to be determined was "sandwiched" by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which has been coupled to a signal generating label.
Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds. Immunochemical assays are reliable when used in the screening of soil for contamination and have been used commercially for the rapid analysis of a variety of compounds, and have been developed to detect a number of different compounds of environmental concern.
In the immunology field the term "hapten" refers to compounds that are unable to directly stimulate antibody production when injected into an animal, but are capable of binding to antibodies if they are produced by an alternate means. Many small molecules do not stimulate the immune system to produce antibodies. Antibodies can be raised, however, that specifically bind to such small molecule haptens. For example, many environmental contaminants, although potentially toxic to humans and animals, do not elicit a strong antibody response.
A "binding assay" is an assay for at least one analyte which may be detected by the formation of a complex between the analyte and an analyte receptor capable of specific interaction with that analyte. The analyte may be haptens, hormones, peptides, proteins, deoxyribonucleic acid (DNA), ribonucleic acids (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific ligand receptor therefor. Binding assays are generally useful for the in vitro determination of the presence and concentration of analyte in body fluids, food products, animal fluids, and environmental samples. For example, the determination of specific hormones, peptides, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition.
Current binding assay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, bioluminescence, fluorescence, fluorescence polarization, a variety of potentiometric and optical biosensor techniques and visual labels such as latex beads, gold particles and carbon black.
There is a continuing need for simple, rapid assays for the qualitative, semi-quantitative, and quantitative determination of such analytes in a sample. Furthermore, in many situations, such assays need to be simple enough to be performed and interpreted by non-technical users.
Binding assays rely on the binding of analyte by analyte receptors to determine the concentrations of analyte in a sample. Analyte-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize analyte receptors in substantial excess over the concentration of analyte to be determined in the assay. Sandwich assays, in which the analyte is detected by binding to two analyte receptors, one analyte receptor labeled to permit detection and a second analyte receptor, frequently bound to a solid phase, to facilitate separation from unbound reagents, such as unbound labeled first analyte receptor, are examples of non-competitive assays.
Competitive assays generally involve a sample suspected of containing analyte, an analyte-analogue conjugate, and the competition of these species for a limited number of binding sites provided by the analyte receptor. Competitive assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of analyte is determined by its effect on the extent of binding between analyte receptor and analyte-conjugate or analyte analogue-conjugate. The signal observed is modulated by the extent of this binding and can be related to the amount of analyte in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive assay in which the analyte analogue conjugate is a analyte analogue-enzyme conjugate and the analyte receptor, in this case an antibody, is capable of binding to either the analyte or the analyte analogue. The binding of the antibody to the analyte analogue-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound analyte and analyte analogue-enzyme conjugate for analyte-receptor binding sites, as the analyte concentration increases the amount of unbound analyte analogue-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
Heterogeneous, competitive assays require a separation of analyte analogue conjugate bound to analyte receptor from the free analyte analogue conjugate and measurements of either the bound or the free fractions. Separation of the bound from the free may be accomplished by removal of the analyte receptor and anything bound to it from the free analyte analogue conjugate by immobilization of the analyte receptor on a solid phase or precipitation. The amount of the analyte analogue conjugate in the bound or the free fraction can then be determined and related to the concentration of the analyte in the sample. Normally the bound fraction is in a convenient form, for example, on a solid phase, so that it can be washed, if necessary, to remove remaining unbound analyte analogue conjugate and the measurement of the bound analyte analogue conjugate or related products is facilitated. The free fraction is normally in a liquid form that is generally inconvenient for measurements. If multiple analytes are being determined in a single assay, the determination of the free fraction of analyte analogue conjugate for each analyte is made impossible if all are mixed in a single liquid unless the responses of the individual analyte analogue conjugates can be distinguished in some manner. However, detecting the free fraction of analyte analogue conjugate in assays that are visually interpreted is a distinct advantage because the density of the color developed in such assays is generally proportional to the analyte concentration over much of the range of analyte concentration.
One method that can be used to detect the free analyte analogue conjugate in a heterogeneous, competitive analyte-receptor assay process is to provide a second, immobilized receptor specific for the analyte on a solid phase so that the analyte analogue conjugate not bound to the first analyte receptor can be bound to the second analyte receptor immobilized on the solid phase.
A serious problem with this approach is that the concentration of analyte in the sample may be several orders of magnitude larger than the concentration of analyte analogue conjugate used in the assay process. Under these circumstances, the analyte and the analyte analogue conjugate compete for the available binding sites on the first analyte receptor resulting in essentially all of the analyte analogue conjugate being free in the assay fluid. When the assay fluid is contacted with the immobilized second receptor, the free analyte and the free analyte analogue conjugate compete for binding sites provided by the second analyte receptor. The excess of free analyte is such that its concentration remains several orders of magnitude larger than that of the free analyte analogue conjugate so that the second analyte receptor binding sites on the solid phase are substantially filled by the analyte. The result of this assay process is that little or no signal may be observed on the solid phase when the concentration of the analyte in the sample is high when in fact the assay should be designed to produce the maximum response for such concentrations of analyte.
In European Patent Application No. 87309724.0, a method is described where the sample suspected of containing the analyte and a analyte analogue conjugate are contacted with a bibulous strip that contains immobilized analyte receptor. When sufficient analyte is present in the sample, free analyte analogue conjugate travels beyond the first immobilized analyte receptor zone and contacts a situs where either analyte receptor or another receptor capable of binding the analyte analogue conjugate is immobilized. If the receptor at the situs is receptor for the analyte, then the problem of competition in the presence of high concentrations of analyte exists as described above. Methods are described where the receptor at the situs is a receptor that binds to a species other than the analyte analogue on the free analyte analogue conjugate so that high concentrations of free analyte do not compete for binding sites at the situs. The use of such receptors at the situs requires the development of additional analyte-receptor pairs for analytes unrelated to the analyte for each analyte to be assayed and restricts these assays to formats where the analyte receptor is immobilized on a solid phase. Under these circumstances the assay of multiple analytes in a single assay becomes complex and difficult to develop.
The method described in U.S. Pat. No. 4,506,009 utilizes an analyte analogue conjugate which has both the analyte analogue and an insolubilizing binding component coupled to the signal development element. An insolubilizing receptor is used to precipitate the free analyte analogue conjugate unless it is sterically hindered by the binding of the antibody specific for analyte to the analyte analogue. This method overcomes some of the deficiencies of the prior art because it provides a method to determine the free fraction of analyte analogue conjugate without interference from the free analyte, but it requires the coupling of two elements, the analyte analogue and the insolubilizing binding component, to the signal development element in such a way that the binding of the antibody to the analyte analogue sterically prevents the binding of the insolubilizing receptor to the insolubilizing binding component. The relative and absolute amounts of the analyte analogue and the insolubilizing binding component that are coupled to the signal development element must be empirically selected to achieve the desired result. The need for such manipulation is both time consuming and may limit the assay performance by restricting the ratio of analyte analogue per signal development element.
U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537, describe a test strip for determining a characteristic of a sample comprising a length of material capable of transporting a developing liquid by capillary action and having zones between its ends for receiving the sample and holding reagents. The strip is used for performing binding assays, particularly those in which a radioisotope is used as a label, such as radioimmunoassays. Minute sample sizes may be used. The strip is capable of application to analytical methods having sensitivities below 0.1 mg/ml.
U.S. Pat. No. 3,893,808, describes a strip of filter paper treated in bands with a chemical reagent, iodine, into which a sample of gasoline suspected of containing lead is wicked from one end and a developing reagent, dithizone, is wicked into the pretreated bands.
U.S. Pat. No. 3,895,914, describes another chemically treated test strip in which chemical reagents are applied in bands or zones on a strip for detecting barbituric acid.
U.S. Pat. No. 4,415,700, describes hydrophilic latex beads consisting of a homo- or co-polymer of monomers containing at least one epoxy group and at least one polymerizable carbon-carbon double bond in each molecule. The method using the particles is a competitive assay wherein labeled first antibody bound analyte and unlabeled first antibody bound analyte compete for binding sites on a particle bound second (anti-first antibody) antibody.
Environmental applications have been explored for the better part of a decade and a number of immunoassay methods have been developed. Most have been used for the detection of herbicides and pesticides in aqueous matrixes. The application of binding assay technology to the testing of solid waste, complex matrixes, and highly lipophilic compounds, has provided unique challenges for the chemist. The feasibility of developing such methods, however, has been demonstrated with immunoassays for single compounds such as Dioxin (see, for example, Vanderlaan, et al. Environmental Toxicology and Chemistry, 7:859-870, 1988; and Stanker, et al., Toxicology, 45: 229-243, 1987).
One of the most serious problems in environmental contamination is the presence of polychlorinated biphenyls (PCBs). PCBs, as commercially available, existed as mixtures of PCB congeners containing various mixtures of 209 different isomeric forms. Toxicological data has indicated that the highly chlorinated PCBs are the most toxic to human health. Because the composition of PCB products varies from individual product to individual product, and even from lot to lot within the same product, binding assay test development is difficult.
Testing is an essential and integral component of all environmental protection and restoration activities. It is the rate limiting element that influences the time, cost, and overall efficiency of project management.
The management of toxic waste sites usually involves a progression through the stages of identification, characterization, remediation and monitoring, with testing being performed during each phase. Reference laboratory methods can effectively identify and quantify unknown compounds in a sample, but become relatively inefficient when used to rapidly locate contamination (i.e., mapping), and assist in remediation and monitoring activities. The complexity of laboratory protocols, and the proximity of the labs to the test site, however, delays the availability of information and increases the cost of obtaining data. The ultimate cost is in the time required by the field crews to collect and test samples for the presence of contaminants. Effective field screening methods can increase the efficiency of the clean-up process by providing an on-site, high-throughput, and cost-effective way to locate contamination and manage its remediation.
The Environmental Protection Agency (EPA) has long promoted and supported the concept of screening methods to supplement laboratory analysis and increase overall efficiency. The need for more effective methods has been recognized in the Superfund Amendments and Reauthorization Act of 1986 which specifies the development and evaluation of alternative time and cost-saving methods that will assist in the eventual remediation of the nations Superfund sites.
Effective field screening methods can increase the efficiency of site management and improve overall data quality when used to supplement the services of regional laboratories. The development of these methods, however, requires a technology that will be compatible with numerous compounds and matrixes and yet be simple, effective and rugged enough to be incorporated into a protocol for use in the field.
Screening methods need to provide fast, simple, cost-effective and reliable information when operated under field conditions. The reagents and equipment should be portable and stable at ambient conditions, and the claims relating to performance should accurately reflect anticipated field use. The methods should be able to rapidly provide an ample quantity of data, and the protocol should be simple to perform and safe to use. Performance characteristics relative to sensitivity, freedom, and correlation to an acceptable reference method should be carefully evaluated. A necessary characteristic of particular significance for screening methods is that they exhibit a very low frequency of false negative results.
Screening methods detect contamination at specified concentrations. The concentration may relate to a hazardous threshold, a clean-up target, or a process-control parameter. The potential implications of false negative data far outweigh those of false positive results. The consequence of a false positive, while a costly problem that needs to be minimized, results in additional testing or treatment. False negative data, however, provides an erroneous perception of a clean site, and may have serious environmental and legal consequences. Safeguards that minimize the incidence of false negative results are imperative.
Thus, a simple binding assay method is needed which will provide reliable, accurate and fast results in the field for a wide range of PCB contaminants (regardless of manufacturer, exact composition or matrix) in a single test using a single antibody. Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping), remediation monitoring, and regulatory compliance.
Similarly, the most widely used products responsible for environmental contamination are refined petroleum products. The contamination of soil and ground water by petroleum products during transport, storage, treatment and disposal is a frequent occurrence. In an attempt to establish the magnitude of the problem, a recent study by the EPA's Underground Storage Tank Program estimated that the U.S. contains approximately 1.4 million underground storage tanks and as many as 400,000 of these tanks may be leaking (see Schwenndeman, et al, in Underground Storage Systems: Leak Detection and Monitoring. Lewis Publishers, Inc., Chelsea, Mich., 1987, 16; Federal Register, Vol. 52, No. 74, 12664 (1987)).
Currently, reference methods for detecting contamination at sites include analysis for benzene, toluene, xylene, ethyl benzene and petroleum hydrocarbons. These methods require laboratory analysis by gas chromatography (GC) or infrared (IR) methods and an extended period of time to obtain the results (see Potter, in Petroleum Contaminated Soils, eds., Calabrese et al., Vol. 2, Lewis Publishers, Chelsea, Mich., 97 (1990)).
While hydrocarbon vapor analyzers can be useful because they provide rapid results, they fail to accurately reflect the amount of hydrocarbons in a soil sample and are unable to detect the more persistent contamination caused by semi-volatile components.
Thus, a binding assay method is needed which will provide reliable accurate and fast results in the field for a wide range of petroleum based contaminants, regardless of manufacturer or exact composition. Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping), remediation, monitoring, and regulatory compliance.
A major disadvantage of prior art positive readout competitive binding assay devices and methods used for detecting small molecules is that the binding affinity of the analyte and its associated binding partner must be sufficiently high to prevent false positives. However, many small molecule binding pairs have insufficient affinity, thus limiting the applicability of this technology to the detection and measurement of small molecules critical for the accurate assessment of environmental quality.
There is also a great need for simple, inexpensive and easy-to-use devices that can be used in doctor offices, at clinics or at home for testing for the presence of therapeutic drugs or hormones in body fluids. Additionally, clinics, emergency medical technicians, corrections facilities, police and firemen require an affordable and easy-to-use device suitable for quickly testing for the presence of drugs of abuse in body fluids outside of a hospital setting.
Thus, a simple, inexpensive, reliable binding assay device and method that is rapid, accurate and precise, and capable of detecting and measuring small molecules and adaptable for field testing is needed.